1. Field of the Invention
The present invention relates to microfluidic thermal control, and in particular, though not exclusively, to thermal control of microfluidic DNA analysis systems using electrical and/or magnetic (hereafter electromagnetic) radiation as an energy source.
2. Related Art
The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. Polymerase chain reaction (PCR) is a well-known technique for amplifying DNA.
With PCR, one can quickly produce millions of copies of DNA starting from a single template DNA molecule. PCR includes a three phase temperature cycle of denaturation of the DNA into single strands, annealing of primers to the denatured strands, and extension of the primers by a thermostable DNA polymerase enzyme. This cycle is repeated a number of times so that at the end of the process there are enough copies to be detected and analyzed. For general details concerning PCR, see Sambrook and Russell, Molecular Cloning—A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2005) and PCR Protocols A Guide to Methods and Applications, M. A. Innis et al., eds., Academic Press Inc. San Diego, Calif. (1990).
Recently, a number of high throughput approaches to performing PCR and other amplification reactions have been developed. In many of these new approaches amplification reactions take place in a microfluidic device. Thermal cycling of the sample for amplification is usually accomplished in one of two methods. In the first method, the sample solution is loaded into the device and the temperature is cycled in time, much like a conventional PCR instrument. In the second method, the sample solution is pumped continuously through spatially varying temperature zones. See, for example, Lagally et al. (Anal Chem 73:565-570 (2001)), Kopp et al. (Science 280:1046-1048 (1998)), Park et al. (Anal Chem 75:6029-6033 (2003)), Hahn et al. (WO 2005/075683), Enzelberger et al. (U.S. Pat. No. 6,960,437) and Knapp et al. (U.S. Patent Application Publication No. 2005/0042639).
Microfluidic systems are systems that have at least one channel through which a fluid may flow, which channel has at least one internal cross-sectional dimension, (e.g., depth, width, length, diameter) that is less than about 1000 micrometers.
There is current market interest in further developing microfluidic genomic sample analysis systems for detecting DNA sequences. The development of these microfluidic systems often entail the various combinations of channel configurations, inlets, outlets, buffer insertion methods, boluses of genomic sample insertion methods, temperature cycling and control methods, and optical analysis methods.
Temperature cycling (thermocyling) and control of samples in a microfluidic system, is an important feature, and varies with particular genomic samples and assays. For example, assays involving denaturation of proteins or thermal cycling reactions during primer extension and nucleic acid amplification reactions require temperature regulation. For example, a typical DNA amplification by polymerase chain reaction (PCR) cycle will cycle the temperature of the genomic sample from about 95° C. for denaturing, to about 55° C. for annealing, then to about 72° C. for extension forming a single PCR cycle. A number of different options are available for achieving such regulation that vary in degree of sophistication.
One specific approach for regulating temperature within the devices is to employ external temperature control sources. Examples of such sources include heating blocks and water baths. Another option is to utilize a heating element such as a resistive heater that can be adjusted to a particular temperature. Such heaters are typically utilized when one seeks to simply maintain a particular temperature. Another suitable temperature controller includes Peltier controllers (e.g., INB Products thermoelectric module model INB-2-(11-4)1.5). This controller is a two stage device capable of heating to 94° C. Such a controller can be utilized to achieve effective thermal cycling or to maintain isothermal incubations at any particular temperature (see discussion in U.S. Application Pub. No. 2004/0115838).
In some devices and applications, heating of a sample directly from a remote heat source has been described, for example, a heating system discussed by Landers (WO 2004/033099 A2), where the heating of a sample is accomplished through the use of energy from a remote heat source, for example infrared (IR). The IR wavelengths are directed to a vessel containing the sample, and because the vessel is made of clear or translucent material, the IR waves act directly on the sample to cause heating of the sample (Landers, pg. 13, 11. 15-24), where heating of the sample is primarily caused by direct action of IR wavelengths on the sample itself. However, for such a system, the absorptive nature of each sample has to be matched to the optical wavelengths of the remote heat source, resulting in a reduced accuracy of temperature stability of a sample depending upon its absorptive characteristics. Decreased temperature stability can result in longer thermal cycling speeds, since it can be difficult to determine the stable temperature at which to plateau, where the thermal cycling speed refers to the time between stabilization from one temperature to another in a heating cycle.
For example, in the PCR process, the thermal cycling speed refers to the time to shift from 95° C. to 55° C. to 72° C. FIG. 1 illustrates a typical variation of temperature zones (A (94° C.), B (52° C.), and C (72° C.)) involved in a conventional PCR process. The faster the thermal cycling speeds and the more accurate the temperature stabilization, the more efficient PCR processes can be performed. Thus, in conventional systems, temperature accuracy and thermal cycling speeds are issues to be resolved.
Additional systems described by Landers et al. (U.S. Pat. No. 6,210,882 and U.S. Pat. No. 6,413,766) are similar to the system described above. For example, they have sample heating occurring by directed sample heating by the IR waves.
In addition to IR remote heating several systems have discussed the use of microwaves to heat the samples directly. For example microwave mediated PCR has been demonstrated using macro volumes with 2.5 mL (Orrling et al., Chem. Comm., 2004, 790-791) and 100 μL reaction volumes (Fermer et al., European Journal of Pharmaceutical Sciences 18:129-132, 2003). In these cases, single-mode microwave cavities were used to deliver microwave power to the sample, and due to the relatively large volumes of liquid being heated, these systems require very high microwave intensities in order to heat the solutions in a reasonable amount of time.
U.S. Pat. No. 6,605,454 to Barenburg et al., discloses a microwave device having a monolithic microwave integrated circuit (MMIC) disposed therein for heating samples introduced into a micro fluidic device and for effecting lysis of cells in the samples by applying microwave radiation. For efficient heating, the patent specifically targets dipole resonance frequency of water in the range of 18 to 26 GHz. This method, thus, is particularly efficient for heating water which is a major component of biological and most chemical systems studied in microfluidic devices. However, the high frequencies required with this approach render the system costly to operate and manufacture.
WO/2006/069305 by Landers et al. discusses a microwave heating system that has a frequency lower than that of the dipole resonance of water. The system described delivers microwave radiation in the frequency range of about 600 MHz-10 GHz. Since these frequencies are lower than the resonance frequency of water, heating efficiency may be improved through matching the impedance of a filled reaction chamber to the transmission line impedance. The microwave heating is controlled by either directly monitoring the solution temperature or, alternatively, remotely monitoring the solution temperature. The system in general delivers microwave radiation to a sample in a micro-area on a microfluidic device, where in one example conductors are placed adjacent to the micro-area for which microwave radiation is desired, where the conductors are close enough to deliver microwave radiation to the sample within the desired micro-area. Thus, as in the other systems discussed above, the system describes direct heating of the sample.